Stretches Of Single-stranded DNA Research Articles

Ultra-deep Coverage Single-molecule R-loop Footprinting Reveals Principles of R-loop Formation

R-loops are a prevalent class of non-B DNA structures that have been associated with both positive and negative cellular outcomes. DNA:RNA immunoprecipitation (DRIP) approaches based on the anti-DNA:RNA hybrid S9.6 antibody revealed that R-loops form dynamically over conserved genic hotspots. We have developed an orthogonal approach that queries R-loops via the presence of long stretches of single-stranded DNA on their looped-out strand. Nondenaturing sodium bisulfite treatment catalyzes the conversion of unpaired cytosines to uracils, creating permanent genetic tags for the position of an R-loop. Long-read, single-molecule PacBio sequencing allows the identification of R-loop ‘footprints’ at near nucleotide resolution in a strand-specific manner on long single DNA molecules and at ultra-deep coverage. Single-molecule R-loop footprinting coupled with PacBio sequencing (SMRF-seq) revealed a strong agreement between S9.6-based and bisulfite-based R-loop mapping and confirmed that R-loops form over genic hotspots, including gene bodies and terminal gene regions. Based on the largest single-molecule R-loop dataset to date, we show that individual R-loops form nonrandomly, defining discrete sets of overlapping molecular clusters that pileup through larger R-loop zones. R-loops most often map to intronic regions and their individual start and stop positions do not match with intron-exon boundaries, reinforcing the model that they form cotranscriptionally from unspliced transcripts. SMRF-seq further established that R-loop distribution patterns are not simply driven by intrinsic DNA sequence features but most likely also reflect DNA topological constraints. Overall, DRIP-based and SMRF-based approaches independently provide a complementary and congruent view of R-loop distribution, consolidating our understanding of the principles underlying R-loop formation.

A structural and dynamic model for the assembly of Replication Protein A on single-stranded DNA

Replication Protein A (RPA), the major eukaryotic single stranded DNA-binding protein, binds to exposed ssDNA to protect it from nucleases, participates in a myriad of nucleic acid transactions and coordinates the recruitment of other important players. RPA is a heterotrimer and coats long stretches of single-stranded DNA (ssDNA). The precise molecular architecture of the RPA subunits and its DNA binding domains (DBDs) during assembly is poorly understood. Using cryo electron microscopy we obtained a 3D reconstruction of the RPA trimerisation core bound with ssDNA (∼55 kDa) at ∼4.7 Å resolution and a dimeric RPA assembly on ssDNA. FRET-based solution studies reveal dynamic rearrangements of DBDs during coordinated RPA binding and this activity is regulated by phosphorylation at S178 in RPA70. We present a structural model on how dynamic DBDs promote the cooperative assembly of multiple RPAs on long ssDNA.

Long repeating (TTAGGG)n single-stranded DNA self-condenses into compact beaded filaments stabilized by G-quadruplex formation

Conformations adopted by long stretches of single-stranded DNA (ssDNA) are of central interest in understanding the architecture of replication forks, R loops, and other structures generated during DNA metabolism in vivo This is particularly so if the ssDNA consists of short nucleotide repeats. Such studies have been hampered by the lack of defined substrates greater than ∼150 nt and the absence of high-resolution biophysical approaches. Here we describe the generation of very long ssDNA consisting of the mammalian telomeric repeat (5'-TTAGGG-3') n , as well as the interrogation of its structure by EM and single-molecule magnetic tweezers (smMT). This repeat is of particular interest because it contains a run of three contiguous guanine residues capable of forming G quartets as ssDNA. Fluorescent-dye exclusion assays confirmed that this G-strand ssDNA forms ubiquitous G-quadruplex folds. EM revealed thick bead-like filaments that condensed the DNA ∼12-fold. The bead-like structures were 5 and 8 nm in diameter and linked by thin filaments. The G-strand ssDNA displayed initial stability to smMT force extension that ultimately released in steps that were multiples ∼28 nm at forces between 6 and 12 pN, well below the >20 pN required to unravel G-quadruplexes. Most smMT steps were consistent with the disruption of the beads seen by EM. Binding by RAD51 distinctively altered the force extension properties of the G-strand ssDNA, suggesting a stochastic G-quadruplex-dependent condensation model that is discussed.

Molecular Genetic Characterization of Mutagenesis Using a Highly Sensitive Single-Stranded DNA Reporter System in Budding Yeast.

Mutations are permanent alterations to the coding content of DNA. They are starting material for the Darwinian evolution of species by natural selection, which has yielded an amazing diversity of life on Earth. Mutations can also be the fundamental basis of serious human maladies, most notably cancers. In this chapter, I describe a highly sensitive reporter system for the molecular genetic analysis of mutagenesis, featuring controlled generation of long stretches of single-stranded DNA in budding yeast cells. This system is ~100- to ~1000-fold more susceptible to mutation than conventional double-stranded DNA reporters, and is well suited for generating large mutational datasets to investigate the properties of mutagens.

Nucleosome-like, Single-stranded DNA (ssDNA)-Histone Octamer Complexes and the Implication for DNA Double Strand Break Repair

Repair of DNA double strand breaks (DSBs) is key for maintenance of genome integrity. When DSBs are repaired by homologous recombination, DNA ends can undergo extensive processing, producing long stretches of single-stranded DNA (ssDNA). In vivo, DSB processing occurs in the context of chromatin, and studies indicate that histones may remain associated with processed DSBs. Here we demonstrate that histones are not evicted from ssDNA after in vitro chromatin resection. In addition, we reconstitute histone-ssDNA complexes (termed ssNucs) with ssDNA and recombinant histones and analyze these particles by a combination of native gel electrophoresis, sedimentation velocity, electron microscopy, and a recently developed electrostatic force microscopy technique, DREEM (dual-resonance frequency-enhanced electrostatic force microscopy). The reconstituted ssNucs are homogenous and relatively stable, and DREEM reveals ssDNA wrapping around histones. We also find that histone octamers are easily transferred in trans from ssNucs to either double-stranded DNA or ssDNA. Furthermore, the Fun30 remodeling enzyme, which has been implicated in DNA repair, binds ssNucs preferentially over nucleosomes, and ssNucs are effective at activating Fun30 ATPase activity. Our results indicate that ssNucs may be a hallmark of processes that generate ssDNA, and that posttranslational modification of ssNucs may generate novel signaling platforms involved in genome stability.

Abstract 1815: ATM inhibition potentiates death of androgen receptor-inactivated prostate cancer cells with telomere dysfunction

Abstract Prostate cancer is the most commonly diagnosed non-skin cancer, and second leading cause of cancer deaths, in American men. The androgen receptor (AR) plays a critical role in the survival of prostate cancer (PCa) cells. Therefore, treatments that inactivate AR, either by decreasing the androgen level or by inhibiting AR directly, are used to treat advanced PCa. However, these treatments are not curative, and disease progression generates castration-resistant prostate cancer (CRPC). Our studies point to a novel role of AR in telomere stability that, in our opinion, can be exploited to enhance the effectiveness of AR-targeted therapies for CRPC. Telomere stability is essential for cell proliferation and survival. We previously reported that AR plays a role in maintaining telomere stability in prostate cancer cells. Since telomere dysfunction activates the DNA damage response (DDR) signaling pathway which can activate cell cycle checkpoints, repair damaged DNA, and promote cell survival, we tested a hypothesis that AR-inactivated telomere dysfunction activates a DDR signaling pathway protein, called ATM (ataxia telangiectasia mutated) kinase, and ATM inhibitor potentiates cell death by averting telomeric DNA repair in AR-inactivated PCa cells. ATM pathway was assessed by the phosphorylation of ATM and its downstream target Chk2 through Western blotting, inhibition of telomere DNA repair was evaluated by counting RPA (DNA replication protein A) foci at damaged telomeres, and the cell viability was determined by PARP cleavage and colony formation assays. We observed that telomere dysfunction by AR-antagonists (Casodex or MDV3100) or AR-siRNA was associated with a dramatic increase in phosphorylation of ATM and Chk2 and the presence of phosphorylated ATM at telomeres, indicating the activation of DNA damage response signaling at telomeres. Moreover, Casodex washout led to the reversal of telomere dysfunction, indicating repair of damaged telomeres. ATM inhibitor blocked ATM phosphorylation and induced PARP cleavage, suggesting that ATM inhibitors induce apoptosis in AR-inactivated cells by blocking the repair of damaged DNA at telomere. This was further corroborated by a conspicuous localization of RPA at damaged telomeres; presence of RPA foci is indicative of unrepaired stretch of single-stranded DNA at telomeres. Finally, colony formation assay revealed a dramatic decrease in the survival of cells co-treated with Casodex and ATM inhibitor as compared with those treated with either Casodex or ATM inhibitor alone. These observations indicate that inhibitors of DDR signaling pathways may offer a unique opportunity to enhance the potency of AR-targeted therapies for the treatment of androgen-sensitive as well as castration- resistant prostate cancer. Citation Format: Sahn-Ho Kim, Vidyavathi Reddy, Min Wu, Evelyn R. Barrack, G. Prem-Veer Reddy. ATM inhibition potentiates death of androgen receptor-inactivated prostate cancer cells with telomere dysfunction. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1815.

Abstract ES01-1: Targeting the DNA damage response in triple negative breast cancer

Abstract Triple negative breast cancer (TNBC) comprises approximately 25 percent of breast cancer deaths. There is a critical need to develop better therapeutic options for TNBC patients. We hypothesize that at least a subset of these cancers are especially dependent on DNA repair mechanisms that are coordinated by the ATR kinase-dependent DNA damage response signaling pathway. This hypothesis has been tested using genetic and chemical genetic approaches. We are working to develop small molecule inhibitors of the ATR pathway, understand how they work, and identify when these targeted therapies may provide clinical benefit. Our studies of an ATR selective inhibitor indicate that it kills cells due to a requirement for ATR signaling to complete DNA replication. ATR inhibition is synergistic with other chemotherapeutic agents such as platinum crosslinking drugs. ATR-inhibitor treated cells accumulate toxic DNA double-strand breaks and large stretches of single-stranded DNA through an active process catalyzed by deregulated nucleases and helicases. Effective biomarkers of ATR inhibition include DNA-PK, H2AX, RPA, and ATR phosphorylation. We have also worked with colleagues at Vanderbilt Medical School to identify additional therapeutic targets and develop small molecule inhibitors. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr ES01-1.

Controlling DNA-end resection: a new task for CDKs

DNA double-strand breaks (DSBs) are repaired by two major pathways: homologous recombination (HR) and non-homologous end-joining (NHEJ). The choice between HR and NHEJ is highly regulated during the cell cycle. DNA-end resection, an evolutionarily conserved process that generates long stretches of single-stranded DNA, plays a critical role in pathway choice, as it commits cells to HR, while, at the same time, suppressing NHEJ. As erroneous DSB repair is a major source of genomic instability-driven tumorigenesis, DNA-end resection factors, and in particular their regulation by post-translational modifications, have become the subject of extensive research over the past few years. Recent work has implicated phosphorylation at S/T-P motifs by cyclin-dependent kinases (CDKs) as a major regulatory mechanism of DSB repair. Intriguingly, CDK activity was found to be critically important for the coordinated and timely execution of DNA-end resection, and key players in this process were subsequently identified as CDK substrates. In this mini review, we provide an overview of the current understanding of how the DNA-end resection machinery in yeast and human cells is controlled by CDK-mediated phosphorylation.

Role of DNA Polymerases in Repeat-Mediated Genome Instability

Expansions of simple DNA repeats cause numerous hereditary diseases in humans. We analyzed the role of DNA polymerases in the instability of Friedreich's ataxia (GAA)(n) repeats in a yeast experimental system. The elementary step of expansion corresponded to ~160 bp in the wild-type strain, matching the size of Okazaki fragments in yeast. This step increased when DNA polymerase α was mutated, suggesting a link between the scale of expansions and Okazaki fragment size. Expandable repeats strongly elevated the rate of mutations at substantial distances around them, a phenomenon we call repeat-induced mutagenesis (RIM). Notably, defects in the replicative DNA polymerases δ and ε strongly increased rates for both repeat expansions and RIM. The increases in repeat-mediated instability observed in DNA polymerase δ mutants depended on translesion DNA polymerases. We conclude that repeat expansions and RIM are two sides of the same replicative mechanism.

Radiation-induced double-strand breaks require ATM but not Artemis for homologous recombination during S-phase

Double-strand breaks (DSBs) are repaired by two distinct pathways, non-homologous end joining (NHEJ) and homologous recombination (HR). The endonuclease Artemis and the PIK kinase Ataxia-Telangiectasia Mutated (ATM), mutated in prominent human radiosensitivity syndromes, are essential for repairing a subset of DSBs via NHEJ in G1 and HR in G2. Both proteins have been implicated in DNA end resection, a mandatory step preceding homology search and strand pairing in HR. Here, we show that during S-phase Artemis but not ATM is dispensable for HR of radiation-induced DSBs. In replicating AT cells, numerous Rad51 foci form gradually, indicating a Rad51 recruitment process that is independent of ATM-mediated end resection. Those DSBs decorated with Rad51 persisted through S- and G2-phase indicating incomplete HR resulting in unrepaired DSBs and a pronounced G2 arrest. We demonstrate that in AT cells loading of Rad51 depends on functional ATR/Chk1. The ATR-dependent checkpoint response is most likely activated when the replication fork encounters radiation-induced single-strand breaks leading to generation of long stretches of single-stranded DNA. Together, these results provide new insight into the role of ATM for initiation and completion of HR during S- and G2-phase. The DSB repair defect during S-phase significantly contributes to the radiosensitivity of AT cells.

Translesion Synthesis of Abasic Sites by Yeast DNA Polymerase ϵ

Studies of replicative DNA polymerases have led to the generalization that abasic sites are strong blocks to DNA replication. Here we show that yeast replicative DNA polymerase epsilon bypasses a model abasic site with comparable efficiency to Pol eta and Dpo4, two translesion polymerases. DNA polymerase epsilon also exhibited high bypass efficiency with a natural abasic site on the template. Translesion synthesis primarily resulted in deletions. In cases where only a single nucleotide was inserted, dATP was the preferred nucleotide opposite the natural abasic site. In contrast to translesion polymerases, DNA polymerase epsilon with 3'-5' proofreading exonuclease activity bypasses only the model abasic site during processive synthesis and cannot reinitiate DNA synthesis. This characteristic may allow other pathways to rescue leading strand synthesis when stalled at an abasic site.

A Bridge Crosses the Active-Site Canyon of the Epstein–Barr Virus Nuclease with DNase and RNase Activities

Epstein–Barr virus, a double-stranded DNA (dsDNA) virus, is a major human pathogen from the herpesvirus family. The nuclease is one of the lytic cycle proteins required for successful viral replication. In addition to the previously described endonuclease and exonuclease activities on single-stranded DNA and dsDNA substrates, we observed an RNase activity for Epstein–Barr virus nuclease in the presence of Mn 2+, giving a possible explanation for its role in host mRNA degradation. Its crystal structure shows a catalytic core of the D-(D/E)XK nuclease superfamily closely related to the exonuclease from bacteriophage lambda with a bridge across the active-site canyon. This bridge may reduce endonuclease activity, ensure processivity or play a role in strand separation of dsDNA substrates. As the DNA strand that is subject to cleavage is likely to make a sharp turn in front of the bridge, endonuclease activity on single-stranded DNA stretches appears to be possible, explaining the cleavage of circular substrates.

Sweet delivery – sugar translocators as ports of entry for antisense oligodeoxynucleotides in plant cells

Antisense oligodeoxynucleotides (ODNs) are short (12-25 nt long) stretches of single-stranded DNA that may be delivered to a cell, where they hybridize to the cognate mRNA in a sequence-specific manner, thereby inhibiting gene expression. Here we used confocal microscopy to monitor the uptake and trafficking of ODNs in barley tissues. We conclude that uptake of ODNs across the plant plasma membrane is mediated by active transport of mono- or disaccharides through sugar translocators. We demonstrate that sugar transport can deliver ODNs to barley seeds, and that this strategy may be employed to suppress gene activity in endosperm cells by antisense ODN inhibition. We further found that sucrose compared favorably with oligofectamine as a vehicle for ODN delivery to human cells in a low-serum environment.

The accessory subunit B of DNA polymerase is required for mitochondrial replisome function

The mitochondrial replication machinery in human cells includes the DNA polymerase γ holoenzyme and the TWINKLE helicase. Together, these two factors form a processive replication machinery, a replisome, which can use duplex DNA as template to synthesize long stretches of single-stranded DNA. We here address the importance of the smaller, accessory B subunit of DNA polymerase γ and demonstrate that this subunit is absolutely required for replisome function. The duplex DNA binding activity of the B subunit is needed for coordination of POLγ holoenzyme and TWINKLE helicase activities at the mtDNA replication fork. In the absence of proof for direct physical interactions between the components of the mitochondrial replisome, these functional interactions may explain the strict interdependence of TWINKLE and DNA polymerase γ for mitochondrial DNA synthesis. Furthermore, mutations in TWINKLE as well as in the catalytic A and accessory B subunits of the POLγ holoenzyme, may cause autosomal dominant progressive external ophthalmoplegia, a disorder associated with deletions in mitochondrial DNA. The crucial importance of the B subunit for replisome function may help to explain why mutations in these three proteins cause an identical syndrome.

A Uracil-DNA Glycosylase Inhibitor Encoded by a Non-uracil Containing Viral DNA

Uracil-DNA glycosylase (UDG) is an enzyme involved in the base excision repair pathway. It specifically removes uracil from both single-stranded and double-stranded DNA. The genome of the Bacillus subtilis phage 29 is a linear double-stranded DNA with a terminal protein covalently linked at each 5'-end. Replication of 29 DNA starts by a protein-priming mechanism and generates intermediates that have long stretches of single-stranded DNA. By using in vivo chemical cross-linking and affinity chromatography techniques, we found that UDG is a cellular target for the early viral protein p56. Addition of purified protein p56 to B. subtilis extracts inhibited the endogenous UDG activity. Moreover, extracts from 29-infected cells were deficient in UDG activity. We suggested that inhibition of the cellular UDG is a defense mechanism developed by 29 to prevent the action of the base excision repair pathway if uracil residues arise in their replicative intermediates. Protein p56 is the first example of a UDG inhibitor encoded by a non-uracil-containing viral DNA.

The Bacillus subtilis Phage φ29 Protein p16.7, Involved in φ29 DNA Replication, Is a Membrane-localized Single-stranded DNA-binding Protein

The functional role of the phi 29-encoded integral membrane protein p16.7 in phage DNA replication was studied using a soluble variant, p16.7A, lacking the N-terminal membrane-spanning domain. Because of the protein-primed mechanism of DNA replication, the bacteriophage phi 29 replication intermediates contain long stretches of single-stranded DNA (ssDNA). Protein p16.7A was found to be an ssDNA-binding protein. In addition, by direct and functional analysis we show that protein p16.7A binds to the stretches of ssDNA of the phi 29 DNA replication intermediates. Properties of protein p16.7A were compared with those of the phi 29-encoded single-stranded DNA-binding protein p5. The results obtained show that both proteins have different, non-overlapping functions. The likely role of p16.7 in attaching phi 29 DNA replication intermediates to the membrane of the infected cell is discussed. Homologues of gene 16.7 are present in phi 29-related phages, suggesting that the proposed role of p16.7 is conserved in this family of phages.

DNA polymerase from Sulfolobus acidocaldarius: Replication at high temperature of long stretches of single-stranded DNA

The activity of a homogeneous DNA polymerase from the thermophilic archaebacterium, Sulfolobus acidocaldarius, on a singly primed, single-stranded recombinant phage M13 DNA has been examined. At the optimal temperature (70 to 75 degrees C) this template is efficiently replicated in ten minutes using a ratio of enzyme molecule to primed-template of 0.8. Analysis of DNA products during the course of polymerization shows that species of quite homogeneous size are observed and that the number of primers extended by the enzyme is constant, whatever the enzyme molecule to primed template ratio is in the range 1/50 to 2, indicating that the 100 x 10(3) Mr DNA polymerase from S. acidocaldarius is randomly recycled on the template molecules. At non-optimal temperature (60 degrees C and 80 degrees C) the distribution of products observed indicated the presence of arrest sequences; some have been shown to be reversible. One of these pausing signals detected at 80 degrees C has been further analysed, and has been found to be DNA sequence-dependent.

Mini-F plasmid-induced SOS signal in Escherichia coli is RecBC dependent.

Dispensable replicons such as F plasmid [95 kilobases (kb)] or its mini-derivatives such as mini-F (9.3 kb) or lambda mini-F efficiently induced cellular SOS genes such as sfiA (sulA) when they were damaged by UV irradiation and then introduced into a recipient bacterium. To generate an SOS signal, UV light-damaged mini-F or mini-F conditional mutants deficient in replication required that the bacterial RecBC enzyme retained some activity different from the nuclease activity that was dispensable. In contrast, UV light-damaged F plasmid produced an SOS signal independently of the activity of the RecBC enzyme and of the expression of the mini-F, -H, and -G proteins. Our findings are consistent with a picture in which the SOS signal is constituted by stretches of single-stranded DNA on a replicon. Moreover, our present data combined with other data previously published lead to the hypothesis that the SOS signal induced by mini-F plasmid is located in trans on the host chromosome, whereas the one generated by UV light-damaged F plasmid is in cis on the transferred DNA.

Branched DNA molecules: Intermediates in T4 recombination

Multiple infection of Escherichia coli with T4 phages defective in both DNA polymerase and ligase leads to the accumulation of DNA structures that are intermediates in recombination. When examined with the electron microscope, up to 25% of these phage DNA molecules contained one or more double-stranded branches of varying lengths and arrangements. Infection at a multiplicity of one failed to give rise to branched molecules. Differences in the frequency of the branched molecules were observed when the infecting phage carried genetic defects in addition to those in polymerase and ligase. DNA of hybrid density extracted from bacteria mixedly-infected with 13C 15N 32P-labeled and 3H-labeled T4 polymerase-ligase mutants was enriched in branched molecules, indicating that the branches were generated in the course of DNA exchange. These findings have suggested a molecular mechanism for recombination based on a process termed “branch migration”, in which there is a concerted dissociation of two sets of homologous parental DNA double helices and reassociation of two sets of hybrid recombinant complements, with a resulting displacement of the branch point sequentially along the interacting DNA molecules. Strand exchange is thus effected without the necessity of exposing long stretches of single-stranded DNA.